Genome of the tardigrade Cucumibius annulatus: functional annotation and specific proteins

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The genomic underpinnings of this remarkable resilience are a subject of intense scientific interest. In this study, we present the first genome assembly and annotation of a tardigrade species Cucumibius annulatus , recently redescribed from North-West Russia. The draft genome was assembled de novo using DNBSEQ sequencing data (PE300 reads), followed by decontamination and annotation. Final assembly has a size of 73 Mb and a BUSCO completeness score of 87.1%. Comparative genomic analysis against three other tardigrade species ( Hypsibius exemplaris , Ramazzottius varieornatus , and Paramacrobiotus metropolitanus ) revealed unique orthologous gene groups in C. annulatus. We identified significant expansions in protein families associated with stress response, including antioxidant enzymes (e.g., Superoxide Dismutase), chaperones (HSP70), and components of the DNA repair and ubiquitin-proteasome systems. Notably, C. annulatus exhibits a pronounced species-specific expansion of signaling receptors, particularly receptor guanylate cyclases and G-protein coupled receptors, as well as extracellular matrix proteins like metalloproteases. These findings suggest that adaptations in signaling pathways and structural proteins, in addition to typical stress-response genes, may play a crucial role in the survival strategies. genome tardigrade stress response comparative genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Tardigrades (phylum Tardigrada) are microscopic invertebrates well known for surviving environmental extremes that are lethal to most animals, including severe desiccation, temperature shocks, and ionizing radiation. A key biological state enabling such endurance is cryptobiosis—most prominently anhydrobiosis—during which metabolism is strongly reduced and organisms can persist in this state for extended periods. The molecular basis of these features is encoded in tardigrade genomes and reflects both conserved eukaryotic stress-response systems and lineage-specific genetic features. [ 1 , 2 , 3 ] Early genome-wide studies highlighted an unexpected range of protective strategies and introduced the idea that tardigrade-unique proteins can directly contribute to cellular protection. A canonical example is the tardigrade damage suppressor protein (Dsup), first characterized in Ramazzottius varieornatus and shown to improve radiotolerance when expressed in human cultured cells. [ 5 ] Later structural work emphasized the intrinsically disordered nature of Dsup and its ability to form a complex with DNA, supporting a model of direct DNA shielding. [ 6 ] Comparative genomics indicates that extremotolerance is not implemented in exactly the same way across all tardigrades. Instead, the sets and copy numbers of tardigrade-specific families (including CAHS/SAHS/MAHS and related abundant heat-soluble proteins) can differ substantially between species, suggesting that closely related phenotypes may rely on partly different molecular “toolkits”. [ 7 , 8 ] Recent evolutionary analyses of desiccation- and temperature-related protein families further support a complex history of stress-associated systems in Tardigrada. [ 9 ] In addition, anhydrobiosis-linked metabolism is not uniform: trehalose production machinery (TPS–TPP) can be absent in some anhydrobiotic lineages due to repeated gene loss and horizontal transfer, implying that trehalose-based strategies are not universal. [ 10 ] Ionizing radiation tolerance provides another clear example of interspecies differences. In Hypsibius exemplaris , radiation tolerance has been linked to strong transcriptional activation of DNA repair pathways (notably BER and NHEJ), and functional experiments implicate at least one core repair factor (XRCC5/Ku80) in survival under irradiation. [ 11 ] Comparative transcriptomics has also identified additional tardigrade-specific irradiation-responsive candidates (e.g., TDR1), supporting the idea that different lineages may rely on partly non-overlapping protective components. [ 12 ] An integrative taxonomic study reclassified Grevenius annulatus as Cucumibius annulatus and established the new genus Cucumibius , supported by detailed morphological evidence including new data on cuticular structure. [ 13 ] As a newly recognized lineage within Isohypsibioidea, C. annulatus provides an opportunity to expand comparative tardigrade genomics and to evaluate how stress-response and cuticle-associated protein repertoires vary across species. In particular, genome-wide analysis allows testing whether commonly discussed extremotolerance-associated families (e.g., CAHS/SAHS/LEA-like protectants, antioxidant enzymes such as SOD, and DNA repair factors) are conserved, expanded, or potentially absent in C. annulatus , and whether other lineage-specific expansions dominate its unique gene space. [ 2 , 3 , 7 , 8 ] Here, we present a de novo genome assembly and structural/functional annotation of Cucumibius annulatus , followed by a comparative proteomic analysis with three reference tardigrades ( Ramazzottius varieornatus , Paramacrobiotus metropolitanus , and Hypsibius exemplaris ). We focus on orthogroups unique to C. annulatus and describe their functional landscape using COG and Pfam annotations, complemented by targeted curation of candidates linked to stress response/DNA repair and to cuticle- and chitin-associated biology. This study provides a genomic resource for a newly established genus and a comparative framework to interpret lineage-specific features that may contribute to stress adaptation and cuticle biology in tardigrades. [ 8 , 13 ] Methods Genome Sequencing and Assembly Specimens of Cucumibius annulatus were collected from the lake Figurnoe, Leningrad region, North-West Russia. DNA isolation was performed with Arcturus PicoPure DNA Extraction Kit (Thermo Fisher Scientific, Massachusetts). The purified DNA was amplified by multiple displacement amplification (MDA) using the REPLI-g Single Cell amplification kit (Qiagen; Thermo Fisher Scientific), according to the manufacturer’s protocol. DNA sequencing was performed at the Core Facility Centre “Biobank” of the Research Park of St. Petersburg University using the MGI DNBSEQ G400 platform (paired-end reads, 300 bp). The resulting reads (56,3 mln) were assembled de novo using SPAdes (v3.15.2) [ 14 ]. Binning and Decontamination Based on the results of the morphological study, the obtained samples were infected with parasites—most likely representatives of Oomycetes. To separate them from the host genome, binning was performed using MaxBin (version 2.2.7), followed by selection of the most plausible bins. The initial assembly was screened for contaminants using BlobToolKit (v2.7.4) [ 8 ]. Contigs identified as originating from bacteria, viruses, or plants based on sequence homology and GC content were removed. Only contigs assigned to Tardigrada, Arthropoda, or those with no hits were retained for the final assembly. The quality and completeness of the final genome assembly were assessed using QUAST (v5.0.2) [ 16 ] and BUSCO (v5.2.2) [ 17 ] against the Eukaryota odb10 database. Gene Prediction and Structural Annotation Gene prediction was performed on the cleaned genome assembly using BRAKER2 (v2.1.6) [ 18 ], which integrates evidence from protein homology and ab initio models from GeneMark-ES/ET [ 19 ] and AUGUSTUS [ 20 ]. Protein evidence for BRAKER was constructed by combining reference proteomes of other tardigrades downloaded from NCBI, including Ramazzottius varieornatus (GCA_001949185.1, Rvar_4.0), Paramacrobiotus metropolitanus (GCF_019649055.1, Prichtersi_v1.0), and Hypsibius exemplaris (GCA_002082055.1, nHd_3.1). Comparative Genomics and Orthogroup Inference To identify orthologous gene families, the predicted proteome of C. annulatus was compared with the proteomes of three other tardigrade species obtained from NCBI: Hypsibius exemplaris (GCA_002082055.1), Ramazzottius varieornatus (GCA_001949185.1), and Paramacrobiotus metropolitanus (GCA_019640555.1). OrthoFinder (v3.1.0) [ 22 ] was used to infer orthogroups (OGs) based on all-vs-all sequence similarity searches performed with DIAMOND [ 16 ]. The resulting orthogroups were analyzed to identify unique, expanded, and conserved gene families in C. annulatus. Functional annotation and COG profiling Functional annotation of the C. annulatus protein set was performed using eggNOG-mapper (emapper-2.1.3) with the eggNOG v5 database (downloaded as emapperdb-5.0.2). [ 23 ] Predicted functions were summarized by COG categories and complemented with KEGG KO and Pfam annotations for downstream enrichment-style interpretation of lineage-specific protein groups. Data processing and visualization Targeted homology searches for trehalose biosynthesis enzymes were performed using DIAMOND (v. 2.1.22; blastp) against the predicted C. annulatus proteome, using multiple reference TPS/TPP sequences ( Saccharomyces cerevisiae Tps1/Tps2; Caenorhabditis elegans TPS-1 and GOB-1; Drosophila melanogaster Tps1; and bacterial OtsA/OtsB from Escherichia coli ). Downstream processing of orthogroup lists and functional annotations (COG/Pfam) as well as plotting figures and interpretation of figures (e.g., Venn diagrams, COG barplots, top-Pfam plots) were performed in R (version used locally: R 4.5.2). Results Analysis of Symbionts and Decontamination We identified nine distinct rRNA sequences corresponding to potential contaminants or symbionts. These included Ferruginibacter spp. (Chitinophagaceae, 98.50% identity for OY970254.1), one Burkholderiaceae species (92.44% for LR585335.1), and Corynebacterium kroppenstedtii (99.89% for CP069509.1), as well as an alphaproteobacterium closely related to endosymbiotic Candidatus Tisiphia (Rickettsiaceae, 99.78% for OZ032159.1). Additionally, we found sequence matching oomycete parasite Haptoglossa zoospora , (98.24% for KT257318.1, Fig. S1 ). The binning procedure successfully separated these non-host sequences from the tardigrade genome, allowing us to focus the subsequent analysis on the host genomic content. We also found SSU rRNA of C. annulatus , and used it for the verification of the taxonomic position of our sample (99.94% for PQ069999.1). Genome Assembly The final genome assembly of Cucumibius annulatus spans 73 Mb, with an N50 of 56 kb and a GC-content of 39.1%. The BUSCO assessment indicated a high level of completeness, with 87.1% of the core eukaryotic genes being present (72.1% for Metazoa dataset). Taxonomic, GC- and coverage-based analyses by BlobToolKit show no signs of contamination. Contig distribution revealed one single “blob” assigned to Tardigrada, Arthropoda and a set of no-hit contigs (Fig. 1 ). The Cucumibius annulatus assembly is within the range of comparable tardigrade genomes in terms of total length (73.0 Mb versus 55.8–170.5 Mb, see Table 1 ), but according to some metrics (N50 = 96.4 kb; L50 = 197), it is more fragmented than the R. varieornatus and P. metropolitanus assemblies. In terms of completeness, our genome assembly is comparable to other tardigrades, but has a lower BUSCO score (Metazoa C = 72.7%), while none of the compared assemblies exceeds 80% (Table 1 ). Table 1 Comparative assembly quality metrics QUAST and BUSCO scores (metazoa_odb10 and eukaryota_odb10) for four tardigrade genome assemblies Data comparison metrics Ramazzottius varieornatus (rvar, GCA_001949185.1) Paramacrobiotus metropolitanus (parmetro, GCA_019649055.1) Hypsibius exemplaris (hypexem, GCA_002082055.1) Cucumibius annulatus (cucumibius, this study) Sequencing technologies Sanger: ABI 3730xl DNA Analyzer; Illumina: Genome Analyzer IIx Illumina HiSeq 4000; Oxford Nanopore MinION PacBio; Illumina HiSeq DNBSEQ Contig/scaffold number (≥ 1000 bp) 200 674 1421 1881 Total length (bp, scaffolds ≥ 1000 bp) 55842812 170501563 104154999 72559506 Average contig/scaffold length (bp) 279214 252970 73297 38575 Longest contig/scaffold length (bp) 9333084 4484719 2115976 785594 N50 (bp) (L50) 4740345 (4) 1033953 (50) 342180 (85) 96445 (197) N90 (bp) (L90) 1295620 (15) 150116 (202) 65573 (343) 21771 (790) auN (bp) 4838420 1319310 467267 146080 GC (%) 47.51 43.46 45.46 39.17 N’s per 100 kbp 750.86 0.06 2058.95 8.70 Total N’s 419303 109 2144499 6310 Percent gaps (%) (N/total) 0.751 0.000 2.059 0.009 BUSCO metazoa (n = 954) C:76.9%[S:75.5%,D:0.6%],F:25.6% C:76.5%[S:73.3%,D:3.2%],F:1.6% C:76.2%[S:74.6%,D:1.6%],F:1.8% C:72.7%[S:71.0%,D:1.8%],F:2.8% BUSCO eukaryota (n = 255) C:91.0%[S:90.2%,D:0.8%],F:2.0% C:90.2%[S:85.5%,D:4.7%],F:2.4%, C:89.4%[S:88.2%,D:1.2%],F:2.4% C:87.1%[S:86.3%,D:0.8%],F:3.5% Comparative Genomics and Orthogroups The BRAKER pipeline predicted a total of 15,875 protein-coding genes in the C. annulatus genome. The OrthoFinder analysis clustered 58,355 proteins from the four tardigrade species into 13,419 orthogroups. A Venn diagram illustrating the distribution of shared and unique orthogroups is shown in Fig. 2 (see detailed list of orthogroups in Table S1 ). A key outcome is the identification of 318 orthogroups unique to C. annulatus . These unique orthogroups collectively contain 1441 C. annulatus proteins, of which 719 received eggNOG functional annotations (COG/Pfam/KO fields available) and were used for quantitative functional profiling (see functional annotation of C. annulatus proteins in Table S2 ). Analysis of specific proteins of C. annulatus -specific groups by COG categories To characterize specific proteins functions, we compared the COG category composition of eggNOG-annotated proteins from C. annulatus -unique orthogroups (n = 719) against the full set of eggNOG-annotated proteins in the C. annulatus proteome (n = 12,042). In the unique set, the most represented categories were T (signal transduction mechanisms; 259 prot., 36.0%), O (posttranslational modification, protein turnover, chaperones; 119, 16.6%) and S (function unknown; 110 prot., 15.3%). Additional contributions were observed for G (carbohydrate transport and metabolism; 39), P (inorganic ion transport and metabolism; 32) and C (energy production and conversion; 29), whereas core “housekeeping” functions, such as translation-related J, were underrepresented among unique proteins (Fig. 3 ). Overall, C. annulatus -specific orthogroups show a pronounced shift toward regulatory/sensory functions (COG T) and protein homeostasis modules (COG O), together with a substantial fraction of proteins that remain functionally uncharacterized (COG S). Pfam domain enrichment among C. annulatus -unique orthogroup proteins Pfam profiling of proteins from unique orthogroups revealed clear domain-level signatures consistent with the COG-based shift toward signaling and extracellular functions (Fig. 4 ). The most frequent domains among unique proteins were: Pkinase (185), Pkinase_Tyr (131), ANF_receptor (120), Guanylate_cyc (116), HNOBA (109). This combination is characteristic of receptor-type guanylate cyclase architectures and strongly suggests a species-specific expansion of cGMP-linked receptor signaling in C. annulatus . Additional enriched domains included 7tm_1 (43) (GPCR-like receptors), and multiple extracellular/ECM-associated modules: Astacin (68), TSP_1 (53), EGF (48), Trypsin (45), ShK (42), CUB (40), as well as Lectin_C (35) and BTB (29). Collectively, these data indicate that the unique orthogroup fraction is dominated by coherent expansions of sensory signaling receptors and extracellular remodeling proteins. To further inspect lineage-specific proteins lacking functional assignments, we focused on the subset classified as COG category S (Function unknown) within the C. annulatus- unique orthogroups. Among these 110 corresponding proteins, all contained identifiable Pfam domains, indicating that they represent structurally defined proteins that included recurrent signatures associated with membrane-associated signaling (e.g., 7tm_1 and XK-related domains), chromatin and transcriptional regulation (e.g., Chromo, SET, PHD and zinc-finger domains), extracellular interactions (e.g., EGF, Lectin_C and CAP domains). Together, these results suggest that the “function unknown” proteins likely represent species-specific regulatory and signaling components contributing to C. annulatus -specific adaptations. Stress-response and repair-related candidates Several unique orthogroups contain proteins classically implicated in stress tolerance and cellular repair (Table 2 ): antioxidant enzymes (SOD-like), chaperones (HSP70-like), selective autophagy receptors (NBR1-like), ubiquitin-mediated proteostasis regulators (BTB/Kelch adaptors), and DDR-associated ubiquitin ligases (e.g., HUWE1-like). Notably, HSP70-family chaperones are generally expected to be conserved; therefore, their assignment to a C. annulatus -specific orthogroup likely reflects orthogroup splitting and/or divergence of particular paralogs rather than true lineage-specificity. Together, these proteins form a coordinated set of mechanisms that support oxidative stress control, proteome maintenance, and DNA damage response pathways. Table 2 Cucumibius annulatus -specific orthogroups with stress-response and repair-related proteins Type Orthogroup (#of proteins) Functional relevance Antioxidant protection (SOD1 (Superoxide dismutase Cu-Zn) and sod) OG0010046 (3) KO: K04565; Pfam: Sod_Cu . Canonical antioxidant enzyme implicated in reactive oxygen species (ROS) detoxification and commonly discussed in the context of stress tolerance. OG0009925 (3) HSP70-like putative paralogs OG0011604 (2) HSP70 is a central molecular chaperone that maintains proteostasis, particularly under stress, by binding unfolded or misfolded proteins, promoting their proper folding, and preventing aggregation. Selective autophagy / protein quality control OG0001225 (9) Presence of NBR1 (selective autophagy receptor; “next to BRCA1 gene”), with domain features consistent with PB1/ZZ/UBA modules. Supports a role in stress-associated clearance of damaged proteins/organelles. Ubiquitination / BTB–Kelch (regulators of protein degradation) OG0000257 (24) Enriched in BTB/BACK/Kelch proteins, which often act as CUL3 E3 ligase adaptors. Expansion may indicate lineage-specific remodeling of ubiquitin-mediated proteostasis and stress signaling. E3 ubiquitin ligases (stress/DDR regulation) OG0010267 (3) RFFL/FEM1-like E3 ubiquitin ligases with ankyrin repeats; regulatory proteins potentially involved in stress and DNA damage response (DDR) pathways DNA repair OG0011414 (2) One protein is annotated as HUWE1 (HECT E3 ubiquitin ligase), which has reported links to ubiquitin-mediated regulation of DNA damage response and/or repair-associated processes Chitin/cuticle-related proteins potentially involved in cuticle remodeling A second set captures candidates potentially involved in tardigrade cuticle organization and remodeling (Table 3 ), including a canonical GH18 chitinase with CBM14 (OG0011702), multiple CBM14 chitin-binding proteins (Peritrophin-A-like signals), multidomain chitin-associated proteins (e.g., CBM14 + LysM + polysaccharide deacetylase-like combinations), and extracellular proteases with chitin-binding modules. This repertoire is consistent with the observed enrichment of extracellular domains (TSP_1/EGF/CUB/Trypsin/Astacin) and supports the hypothesis of a dynamically regulated and lineage-specific extracellular/cuticular toolkit in C. annulatus . [ 13 ] Table 3 Chitin-related proteins potentially involved in cuticle organization and remodeling in Cucumibius annulatus Type Orthogroup (#of proteins) Pfam / domains KO / annotation clue Role in tardigrade cuticle biology Chitinases (chitin degradation / remodeling) OG0011702 (2) Glyco_hydro_18 + CBM_14 KO: K01183 (chitinase) Canonical GH18 chitinase with a chitin-binding module; likely involved in remodeling of chitin-containing structures (e.g., cuticle) Chitinase-like (atypical domain architecture; requires validation) OG0010212 (1) Glyco_hydro_19 Chitinase class I Chitinase-like protein; GH19 is unusual for animals OG0011447 (1) Chitin-binding proteins (structural/extracellular components) OG0001235 (9) CBM_14; sometimes Chitin_bind_4 (e.g., g15380.t1; g1810.t1) Peritrophin-A / chitin binding Candidate chitin-binding extracellular/cuticular proteins that may contribute to cuticle architecture and mechanical properties Chitin binding + chitin metabolic process (multidomain) OG0011703 (2) CBM_14, LysM, Polysacc_deac_1 (+ Cu-binding_MopE) chitin binding; chitin metabolic process Multidomain candidate involved in chitin-associated metabolism or matrix interactions; may modulate chitin structure (e.g., via polysaccharide deacetylase-like activity) Proteases with chitin-binding modules (extracellular remodeling) OG0000353 (19) Chitin_bind_4, NIDO, Somatomedin_B, Trypsin protease-like Secreted/ECM-associated protease architecture with chitin-binding features; potentially participates in cuticle/ECM remodeling (protein component turnover and matrix processing) Protease with CBM_14 (candidate cuticle remodeling factor) OG0011230 (1) CBM_14, Trypsin (+ PAP2, RED_C/RED_N, RRM_1) protease-like Candidate proteolytic component with chitin-binding module; may contribute to remodeling of cuticle-associated proteins (domain combination suggests complex regulation) Chitin synthase OG0007650 (1) Chitin_synth_1,Chitin_synth_1N,Chitin_synth_2,EGF,F5_F8_type_C,FYVE_2,Glyco_hydro_47,Glyco_trans_2_3,I-set,JAB,Myosin_head,OTU,PKD_channel,Rab_eff_C,SAM_1,SAM_2,SRCR,TSP_1,UQ_con ko:K00698 Within two orthogroups, they are also found in other species: rvar (1 ), parmetro (3), hypexem (2) OG0003428 (2) Discussion Lineage-specific expansions and molecular adaptations Extensive expansion of receptor-associated signaling proteins in C. annulatus genome suggest that it invests heavily in membrane-associated sensing and regulatory networks, potentially enhancing the detection of environmental changes and coordination of physiological transitions associated with dehydration and cryptobiosis. A second major lineage-specific theme is the expansion of extracellular and matrix-associated proteins. Together with the presence of chitin- and cuticle-related proteins (including GH18 chitinases with CBM14 and multiple chitin-binding proteins), these findings suggest that C. annulatus possesses an expanded toolkit for cuticle remodeling and extracellular structural maintenance, processes that are critical during molting and may contribute to mechanical stability and barrier function during desiccation. Cucumibius annulatus is the first species of Isohypsibioidea for which a genome has been assembled. Based on rRNA tree topology, this superfamily is a basal lineage within Parachela [ 13 ]. C. annulatus is strictly freshwater, does not occur in terrestrial habitats, and most likely cannot tolerate drying out. Phylogenetic placement raises the possibility that the prominent expansions we observe—especially in receptor-like signaling proteins and chitin/cuticle-associated genes—reflect either retained ancestral features or lineage-specific adaptations linked to the unusually complex cuticle morphology of C. annulatus. We additionally identified a lineage-specific expansion of globin-domain proteins (OG0000357; 19 proteins). Beyond classical roles in oxygen transport, globins in small invertebrates are often implicated in redox balance and stress physiology. In the context of cryptobiotic cycles and fluctuating oxygen availability, an expanded globin repertoire may contribute to oxygen homeostasis and mitigation of oxidative stress during entry into and recovery from dormant states. Conserved maintenance systems versus lineage-specific solutions Despite the pronounced lineage-specific expansions, C. annulatus retains a conserved core of stress-response and maintenance pathways typical for tardigrades. Comparative analysis revealed the presence of key DNA repair enzymes (including XPF-, MUS81-, FEN1-, DNA2-, and EXO1-like proteins), as well as a well-developed RNA surveillance and RNAi machinery (exosome components, mRNA decapping and degradation factors, and Dicer- and MUT-7-like proteins), supporting an active model of genome and transcriptome maintenance under stress. Importantly, C. annulatus shares canonical tardigrade heat-soluble protein families with R. varieornatus , including 16 CAHS, 13 SAHS and 3 LEA proteins, indicating that the classical stress-protection toolkit is conserved and may support basal stress tolerance. At the same time, the retention of CAHS, SAHS (and LEA) may be biologically relevant beyond anhydrobiosis, as CAHS proteins have been implicated in protection against multiple stress types, including cold stress/cryobiosis. This is consistent with the distribution of C. annulatus in boreal and mountainous regions (so called Arctic-alpine areal type) where freshwater habitats can freeze extensively. In contrast, we did not detect convincing Dsup-like homologs in the predicted C. annulatus proteome using similarity searches with Dsup from R. varieornatus , suggesting that a Dsup-based DNA-shielding mechanism is either absent or too divergent to be recovered by homology-based approaches [ 5 , 6 ]. In addition, we detect convincing candidates for the trehalose biosynthesis machinery (TPS–TPP) in the predicted C. annulatus proteome − 9 proteins with trehalose phosphatase domains (Trehalose_PPase). This number is consistent with the amount of trehalose biosynthesis pathway members among other tardigrade species in the study. Trehalose can act as a cryoprotectant and chemical chaperone. Overwintering under ice can impose hypoxic conditions. The lineage-specific expansion of globin-domain proteins in C. annulatus therefore may be interpreted not only in terms of oxidative stress control, but also as a potential adaptation supporting oxygen homeostasis during seasonal oxygen limitation. However, in contrast to R. varieornatus , the dominant lineage-specific trend in C. annulatus is not the continued expansion of the heat-soluble proteins, but rather large-scale diversification of signaling receptors and extracellular proteins. These results suggest that C. annulatus achieves extremotolerance through a combination of conserved protective mechanisms and lineage-specific regulatory and structural adaptations. In particular, the expansion of signaling receptors and extracellular/structural protein families distinguishes C. annulatus from other sequenced tardigrades and may represent an important, previously underappreciated component of tardigrade adaptation. Conclusion In this study, we provide the first genome assembly, structural annotation and comparative orthogroup-based analysis for the recently established tardigrade genus Cucumibius and its species Cucumibius annulatus . The genome-wide comparison against three reference tardigrades indicates 318 orthogroups unique to C. annulatus species, with a functional profile strongly shifted toward signal transduction and protein turnover/proteostasis. This pattern suggests that C. annulatus combines a conserved “core” of stress-response mechanisms with pronounced lineage-specific expansions that likely affect how environmental cues are sensed and how extracellular structures are maintained. Declarations Ethical Approval Not applicable Clinical trial number Not applicable Consent to participate Not applicable Consent to publish All authors agreed with the content and that all gave explicit consent to submit. They obtained consent from the responsible authorities at the institute where the work has been carried out Data Availability Statement Genome assembly was deposited as NCBI BioSample, accession number SAMN55414403 Authors Contributions All authors contributed to the study conception and design. Sample collection and material preparation were performed by Denis Tumanov. Preparation of material for library construction and sequencing was performed by Elena Nassonova. Data collection and analysis were performed by Daria Makarova and Mikhail Rayko. The first draft of the manuscript was written by Daria Makarova and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This study was supported by the Russian Science Foundation: project no. 25-74-20033 (sample collection, morphological description, and species identification) and project no. 23–74-00071 (DNA extraction, whole genome amplification, sequencing, metagenome assembly, analysis of parasite and host genomes, bioinformatic data processing). Competing Interests The authors declare no conflicts of interest. Acknowledgements This study utilised equipment of the Core Facility Centres ‘Biobank’, ‘Development of Molecular and Cell Technologies’ and ‘Culturing of microorganisms’ of the Research Park of Saint Petersburg University References Guidetti R, Jönsson KI (2002) Long-term anhydrobiotic survival in semi-terrestrial tardigrades. J Zool 257(2):181–187 Møbjerg N, Halberg KA, Jørgensen A, Persson D, Bjørn M, Ramløv H, Kristensen RM (2011) Survival in extreme environments by tardigrades. J Exp Biol 214(Pt 1):1–11 Arakawa K (2022) Examples of Extreme Survival: Tardigrade Genomics and Molecular Anhydrobiology. Annu Rev Anim Biosci 10:17–37. 10.1146/annurev-animal-021419-083711 Carrero D, Pérez-Silva JG, Quesada V, López-Otín C (2019) Differential mechanisms of tolerance to extreme environmental conditions in tardigrades. Sci Rep 9:14938. 10.1038/s41598-019-51471-8 Hashimoto T, Horikawa DD, Saito Y et al (2016) Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun 7:12808. 10.1038/ncomms12808 Zarubin M, Murugova T, Ryzhykau Y, Ivankov O, Uversky VN, Kravchenko E (2024) Structural study of the intrinsically disordered tardigrade damage suppressor protein (Dsup) and its complex with DNA. Sci Rep 14:74335. 10.1038/s41598-024-74335-2 Boothby TC, Tapia H, Brozena AH et al (2017) Tardigrades use intrinsically disordered proteins to survive desiccation. 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Curr Biol 34(9):1819–1830e6. 10.1016/j.cub.2024.03.019 Anoud M, Delagoutte E, Helleu Q et al (2024) Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation. eLife 13:RP92621. 10.7554/eLife.92621 Tumanov DV, Shunatova NN, Fedyuk KA (2025) Integrative description of Grevenius annulatus … leads to the institution of a new genus. Zoolog Scr 54(2):232–252. 10.1111/zsc.12703 Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477 Challis R, Richards E, Rajan J, Cochrane G, Blaxter M (2020) BlobToolKit—Interactive quality assessment of genome assemblies. G3: Genes Genomes Genet 10(4):1361–1374 Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST: quality assessment tool for genome assemblies. Bioinformatics 29(8):1072–1075 Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM (2021) BUSCO update… Molecular Biology Evolution 38(10):4647–4654 Brůna T, Hoff KJ, Lomsadze A, Stanke M, Borodovsky M (2021) BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP + and AUGUSTUS supported by a protein database. NAR Genomics Bioinf 3(1):lqaa108. https://doi.org/10.1093/nargab/lqaa108 Lomsadze A, Ter-Hovhannisyan V, Chernoff YO, Borodovsky M (2005) Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res 33(20):6494–6506. https://doi.org/10.1093/nar/gki937 Stanke M, Keller O, Gunduz I, Hayes A, Waack S, Morgenstern B (2006) AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res 34(suppl2):W435–W439. https://doi.org/10.1093/nar/gkl200 Emms DM, Kelly S (2019) OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 20:238. https://doi.org/10.1186/s13059-019-1832-y Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12(1):59–60 Huerta-Cepas J, Forslund K, Coelho LP, Szklarczyk D, Jensen LJ, von Mering C, Bork P (2017) Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol Biol Evol 34(8):2115–2122. https://doi.org/10.1093/molbev/msx148 Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx Table S1.xls. Detailed list of orthogroups from four tardigrade species in the study, obtained by OrthoFinder TableS2.xlsx Table S2.xls.Functional annotation of proteomes from four species in the study, obtained by eggNOG-mapper. SupplementalMaterials.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 11 Apr, 2026 Reviews received at journal 08 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers invited by journal 03 Mar, 2026 Editor assigned by journal 25 Feb, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 24 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8958382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600095094,"identity":"33e1922b-0cc5-48e0-a73c-ba522880d0ac","order_by":0,"name":"Daria Makarova","email":"","orcid":"","institution":"ITMO University","correspondingAuthor":false,"prefix":"","firstName":"Daria","middleName":"","lastName":"Makarova","suffix":""},{"id":600095095,"identity":"fab26490-cde0-45f1-a632-399c72150b8f","order_by":1,"name":"Denis Tumanov","email":"","orcid":"","institution":"St Petersburg University","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Tumanov","suffix":""},{"id":600095096,"identity":"e95b6c69-c2d3-4234-8db0-00d33c6ac00e","order_by":2,"name":"Elena Nassonova","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Nassonova","suffix":""},{"id":600095097,"identity":"9f4d626f-169e-447e-ab69-49cf6cc97aab","order_by":3,"name":"Mikhail Rayko","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACZjYILcHAfABEyZCihS0BRPEQYQ1cC48BiCasRbedLfEB4x67xJntPZ9f3aix4GFgP3x0Az4tZofZDhswPEtOnM1zdpt1zjGgw3jS0m7g18LeJsFwgDlxnkTuNuMcNqAWCR4zQlrafzAcqAdqyXlmnPOPKC1sxxgYDhxOnC2Rw/w4t404LckSCQeOG8/sOWbGnNsnwcNG0C/njxl++HCgWnbG8ebHn3O+1cnxsx8+hlcLGCQwMDg2AGNIAsRhI6gcCuyBmPkDsapHwSgYBaNgZAEAFcJF9MPqdTEAAAAASUVORK5CYII=","orcid":"","institution":"ITMO University","correspondingAuthor":true,"prefix":"","firstName":"Mikhail","middleName":"","lastName":"Rayko","suffix":""}],"badges":[],"createdAt":"2026-02-24 14:09:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8958382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8958382/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104082784,"identity":"3407f6e2-8695-4c0e-b6d2-1d12cb577cfa","added_by":"auto","created_at":"2026-03-06 14:42:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91080,"visible":true,"origin":"","legend":"\u003cp\u003eBlobToolKit GC–coverage plot of assembled contigs with taxonomic assignments. The dominant cluster corresponds to the target Tardigrada bin; non-target contigs were removed during decontamination.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/a4f9cd893ba80c3ba158dc3f.png"},{"id":104082792,"identity":"890a4a7f-fa96-44e0-9de6-8c80f1a7ca95","added_by":"auto","created_at":"2026-03-06 14:42:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145005,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram showing the intersection of orthogroups between four species of tardigrades: \u003cem\u003eRamazzottius varieornatus \u003c/em\u003e(rvar), \u003cem\u003eParamacrobiotus metropolitanus\u003c/em\u003e (parmetro), \u003cem\u003eHypsibius exemplaris\u003c/em\u003e (hypexem), and \u003cem\u003eCucumibius annulatu\u003c/em\u003es (cucumibius). The numbers in the sectors correspond to the number of orthogroups that are common or unique to the respective species.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/83f1f567efafddcb45201ee3.png"},{"id":104082783,"identity":"d59a5922-446f-4c18-8a7c-f8bf3b644af6","added_by":"auto","created_at":"2026-03-06 14:42:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":294162,"visible":true,"origin":"","legend":"\u003cp\u003eCOG category distribution in \u003cem\u003eCucumibius annulatus \u003c/em\u003eproteome: unique orthogroup proteins vs all eggNOG-annotated proteins.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/8e1e69bb09b61d46c747b1ab.png"},{"id":104082791,"identity":"f1008542-f7a9-413f-886a-1dbffd73c91f","added_by":"auto","created_at":"2026-03-06 14:42:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58992,"visible":true,"origin":"","legend":"\u003cp\u003eTop-20 Pfam domains among proteins from \u003cem\u003eCucumibius annulatus\u003c/em\u003e-specific orthogroups (eggNOG-mapper Pfam assignments). Numbers indicate domain counts.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/cbad601c1a8b82f80d0ddb86.png"},{"id":104808364,"identity":"879caae8-8c5b-487f-b8e1-d76691de4068","added_by":"auto","created_at":"2026-03-17 12:36:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1973238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/188f06f4-e8af-4d86-9adc-91c7df57986a.pdf"},{"id":104082801,"identity":"68b47b79-6d35-4bfd-ae4a-052bbb14d9a1","added_by":"auto","created_at":"2026-03-06 14:42:13","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":590393,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1.xls.\u003c/strong\u003e \u0026nbsp;Detailed list of orthogroups from four tardigrade species in the study, obtained by OrthoFinder\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/e994dba4e1bda1665a31b9cc.xlsx"},{"id":104082787,"identity":"7c2fb6a1-c56a-448b-98c0-3f0057c6c12c","added_by":"auto","created_at":"2026-03-06 14:42:11","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6597144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2.xls.\u003c/strong\u003eFunctional annotation of proteomes from four species in the study, obtained by eggNOG-mapper.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/003a80b6cb8e61f6df7497d2.xlsx"},{"id":104082790,"identity":"836d89df-47e6-47bf-8677-1ec26f4dc887","added_by":"auto","created_at":"2026-03-06 14:42:12","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":475980,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8958382/v1/9b9c14d0097f3c41926431f4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome of the tardigrade Cucumibius annulatus: functional annotation and specific proteins","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTardigrades (phylum Tardigrada) are microscopic invertebrates well known for surviving environmental extremes that are lethal to most animals, including severe desiccation, temperature shocks, and ionizing radiation. A key biological state enabling such endurance is cryptobiosis\u0026mdash;most prominently anhydrobiosis\u0026mdash;during which metabolism is strongly reduced and organisms can persist in this state for extended periods. The molecular basis of these features is encoded in tardigrade genomes and reflects both conserved eukaryotic stress-response systems and lineage-specific genetic features. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eEarly genome-wide studies highlighted an unexpected range of protective strategies and introduced the idea that tardigrade-unique proteins can directly contribute to cellular protection. A canonical example is the tardigrade damage suppressor protein (Dsup), first characterized in \u003cem\u003eRamazzottius varieornatus\u003c/em\u003e and shown to improve radiotolerance when expressed in human cultured cells. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Later structural work emphasized the intrinsically disordered nature of Dsup and its ability to form a complex with DNA, supporting a model of direct DNA shielding. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eComparative genomics indicates that extremotolerance is not implemented in exactly the same way across all tardigrades. Instead, the sets and copy numbers of tardigrade-specific families (including CAHS/SAHS/MAHS and related abundant heat-soluble proteins) can differ substantially between species, suggesting that closely related phenotypes may rely on partly different molecular \u0026ldquo;toolkits\u0026rdquo;. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Recent evolutionary analyses of desiccation- and temperature-related protein families further support a complex history of stress-associated systems in Tardigrada. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] In addition, anhydrobiosis-linked metabolism is not uniform: trehalose production machinery (TPS\u0026ndash;TPP) can be absent in some anhydrobiotic lineages due to repeated gene loss and horizontal transfer, implying that trehalose-based strategies are not universal. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIonizing radiation tolerance provides another clear example of interspecies differences. In \u003cem\u003eHypsibius exemplaris\u003c/em\u003e, radiation tolerance has been linked to strong transcriptional activation of DNA repair pathways (notably BER and NHEJ), and functional experiments implicate at least one core repair factor (XRCC5/Ku80) in survival under irradiation. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Comparative transcriptomics has also identified additional tardigrade-specific irradiation-responsive candidates (e.g., TDR1), supporting the idea that different lineages may rely on partly non-overlapping protective components. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAn integrative taxonomic study reclassified \u003cem\u003eGrevenius annulatus\u003c/em\u003e as \u003cem\u003eCucumibius annulatus\u003c/em\u003e and established the new genus \u003cem\u003eCucumibius\u003c/em\u003e, supported by detailed morphological evidence including new data on cuticular structure. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] As a newly recognized lineage within Isohypsibioidea, \u003cem\u003eC. annulatus\u003c/em\u003e provides an opportunity to expand comparative tardigrade genomics and to evaluate how stress-response and cuticle-associated protein repertoires vary across species. In particular, genome-wide analysis allows testing whether commonly discussed extremotolerance-associated families (e.g., CAHS/SAHS/LEA-like protectants, antioxidant enzymes such as SOD, and DNA repair factors) are conserved, expanded, or potentially absent in \u003cem\u003eC. annulatus\u003c/em\u003e, and whether other lineage-specific expansions dominate its unique gene space. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eHere, we present a de novo genome assembly and structural/functional annotation of \u003cem\u003eCucumibius annulatus\u003c/em\u003e, followed by a comparative proteomic analysis with three reference tardigrades (\u003cem\u003eRamazzottius varieornatus\u003c/em\u003e, \u003cem\u003eParamacrobiotus metropolitanus\u003c/em\u003e, and \u003cem\u003eHypsibius exemplaris\u003c/em\u003e). We focus on orthogroups unique to \u003cem\u003eC. annulatus\u003c/em\u003e and describe their functional landscape using COG and Pfam annotations, complemented by targeted curation of candidates linked to stress response/DNA repair and to cuticle- and chitin-associated biology. This study provides a genomic resource for a newly established genus and a comparative framework to interpret lineage-specific features that may contribute to stress adaptation and cuticle biology in tardigrades. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenome Sequencing and Assembly\u003c/h2\u003e \u003cp\u003eSpecimens of \u003cem\u003eCucumibius annulatus\u003c/em\u003e were collected from the lake Figurnoe, Leningrad region, North-West Russia. DNA isolation was performed with Arcturus PicoPure DNA Extraction Kit (Thermo Fisher Scientific, Massachusetts). The purified DNA was amplified by multiple displacement amplification (MDA) using the REPLI-g Single Cell amplification kit (Qiagen; Thermo Fisher Scientific), according to the manufacturer\u0026rsquo;s protocol. DNA sequencing was performed at the Core Facility Centre \u0026ldquo;Biobank\u0026rdquo; of the Research Park of St. Petersburg University using the MGI DNBSEQ G400 platform (paired-end reads, 300 bp). The resulting reads (56,3 mln) were assembled de novo using SPAdes (v3.15.2) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBinning and Decontamination\u003c/h3\u003e\n\u003cp\u003eBased on the results of the morphological study, the obtained samples were infected with parasites\u0026mdash;most likely representatives of Oomycetes. To separate them from the host genome, binning was performed using MaxBin (version 2.2.7), followed by selection of the most plausible bins. The initial assembly was screened for contaminants using BlobToolKit (v2.7.4) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Contigs identified as originating from bacteria, viruses, or plants based on sequence homology and GC content were removed. Only contigs assigned to Tardigrada, Arthropoda, or those with no hits were retained for the final assembly. The quality and completeness of the final genome assembly were assessed using QUAST (v5.0.2) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and BUSCO (v5.2.2) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] against the Eukaryota odb10 database.\u003c/p\u003e\n\u003ch3\u003eGene Prediction and Structural Annotation\u003c/h3\u003e\n\u003cp\u003eGene prediction was performed on the cleaned genome assembly using BRAKER2 (v2.1.6) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], which integrates evidence from protein homology and ab initio models from GeneMark-ES/ET [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and AUGUSTUS [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Protein evidence for BRAKER was constructed by combining reference proteomes of other tardigrades downloaded from NCBI, including \u003cem\u003eRamazzottius varieornatus\u003c/em\u003e (GCA_001949185.1, Rvar_4.0), \u003cem\u003eParamacrobiotus metropolitanus\u003c/em\u003e (GCF_019649055.1, Prichtersi_v1.0), and \u003cem\u003eHypsibius exemplaris\u003c/em\u003e (GCA_002082055.1, nHd_3.1).\u003c/p\u003e\n\u003ch3\u003eComparative Genomics and Orthogroup Inference\u003c/h3\u003e\n\u003cp\u003eTo identify orthologous gene families, the predicted proteome of \u003cem\u003eC. annulatus\u003c/em\u003e was compared with the proteomes of three other tardigrade species obtained from NCBI: \u003cem\u003eHypsibius exemplaris\u003c/em\u003e (GCA_002082055.1), \u003cem\u003eRamazzottius varieornatus\u003c/em\u003e (GCA_001949185.1), and \u003cem\u003eParamacrobiotus metropolitanus\u003c/em\u003e (GCA_019640555.1). OrthoFinder (v3.1.0) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was used to infer orthogroups (OGs) based on all-vs-all sequence similarity searches performed with DIAMOND [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The resulting orthogroups were analyzed to identify unique, expanded, and conserved gene families in \u003cem\u003eC. annulatus.\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eFunctional annotation and COG profiling\u003c/h3\u003e\n\u003cp\u003eFunctional annotation of the \u003cem\u003eC. annulatus\u003c/em\u003e protein set was performed using eggNOG-mapper (emapper-2.1.3) with the eggNOG v5 database (downloaded as emapperdb-5.0.2). [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Predicted functions were summarized by COG categories and complemented with KEGG KO and Pfam annotations for downstream enrichment-style interpretation of lineage-specific protein groups.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData processing and visualization\u003c/h2\u003e \u003cp\u003eTargeted homology searches for trehalose biosynthesis enzymes were performed using DIAMOND (v. 2.1.22; blastp) against the predicted \u003cem\u003eC. annulatus\u003c/em\u003e proteome, using multiple reference TPS/TPP sequences (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e Tps1/Tps2; \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e TPS-1 and GOB-1; \u003cem\u003eDrosophila melanogaster\u003c/em\u003e Tps1; and bacterial OtsA/OtsB from \u003cem\u003eEscherichia coli\u003c/em\u003e). Downstream processing of orthogroup lists and functional annotations (COG/Pfam) as well as plotting figures and interpretation of figures (e.g., Venn diagrams, COG barplots, top-Pfam plots) were performed in R (version used locally: R 4.5.2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Symbionts and Decontamination\u003c/h2\u003e \u003cp\u003eWe identified nine distinct rRNA sequences corresponding to potential contaminants or symbionts. These included \u003cem\u003eFerruginibacter\u003c/em\u003e spp. (Chitinophagaceae, 98.50% identity for OY970254.1), one Burkholderiaceae species (92.44% for LR585335.1), and \u003cem\u003eCorynebacterium kroppenstedtii\u003c/em\u003e (99.89% for CP069509.1), as well as an alphaproteobacterium closely related to endosymbiotic Candidatus \u003cem\u003eTisiphia\u003c/em\u003e (Rickettsiaceae, 99.78% for OZ032159.1). Additionally, we found sequence matching oomycete parasite \u003cem\u003eHaptoglossa zoospora\u003c/em\u003e, (98.24% for KT257318.1, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The binning procedure successfully separated these non-host sequences from the tardigrade genome, allowing us to focus the subsequent analysis on the host genomic content. We also found SSU rRNA of \u003cem\u003eC. annulatus\u003c/em\u003e, and used it for the verification of the taxonomic position of our sample (99.94% for PQ069999.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenome Assembly\u003c/h2\u003e \u003cp\u003eThe final genome assembly of \u003cem\u003eCucumibius annulatus\u003c/em\u003e spans 73 Mb, with an N50 of 56 kb and a GC-content of 39.1%. The BUSCO assessment indicated a high level of completeness, with 87.1% of the core eukaryotic genes being present (72.1% for Metazoa dataset). Taxonomic, GC- and coverage-based analyses by BlobToolKit show no signs of contamination. Contig distribution revealed one single \u0026ldquo;blob\u0026rdquo; assigned to Tardigrada, Arthropoda and a set of no-hit contigs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003eCucumibius annulatus\u003c/em\u003e assembly is within the range of comparable tardigrade genomes in terms of total length (73.0 Mb versus 55.8\u0026ndash;170.5 Mb, see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but according to some metrics (N50\u0026thinsp;=\u0026thinsp;96.4 kb; L50\u0026thinsp;=\u0026thinsp;197), it is more fragmented than the \u003cem\u003eR. varieornatus\u003c/em\u003e and \u003cem\u003eP. metropolitanus\u003c/em\u003e assemblies. In terms of completeness, our genome assembly is comparable to other tardigrades, but has a lower BUSCO score (Metazoa C\u0026thinsp;=\u0026thinsp;72.7%), while none of the compared assemblies exceeds 80% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative assembly quality metrics QUAST and BUSCO scores (metazoa_odb10 and eukaryota_odb10) for four tardigrade genome assemblies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eData comparison metrics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRamazzottius varieornatus\u003c/em\u003e (rvar, GCA_001949185.1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eParamacrobiotus metropolitanus\u003c/em\u003e (parmetro, GCA_019649055.1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHypsibius exemplaris\u003c/em\u003e (hypexem, GCA_002082055.1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCucumibius annulatus\u003c/em\u003e (cucumibius, this study)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSequencing technologies\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanger: ABI 3730xl DNA Analyzer; Illumina: Genome Analyzer IIx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIllumina HiSeq 4000; Oxford Nanopore MinION\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePacBio; Illumina HiSeq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDNBSEQ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eContig/scaffold number (\u0026ge;\u0026thinsp;1000 bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e674\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1881\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal length (bp, scaffolds\u0026thinsp;\u0026ge;\u0026thinsp;1000 bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55842812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e170501563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e104154999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72559506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAverage contig/scaffold length (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e279214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e252970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73297\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38575\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLongest contig/scaffold length (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9333084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4484719\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2115976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e785594\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN50 (bp) (L50)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4740345 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1033953 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e342180 (85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96445 (197)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN90 (bp) (L90)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1295620 (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150116 (202)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65573 (343)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21771 (790)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eauN (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4838420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1319310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e467267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e146080\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGC (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN\u0026rsquo;s per 100 kbp\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e750.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2058.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal N\u0026rsquo;s\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e419303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2144499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6310\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePercent gaps (%) (N/total)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBUSCO metazoa\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;954)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC:76.9%[S:75.5%,D:0.6%],F:25.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC:76.5%[S:73.3%,D:3.2%],F:1.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC:76.2%[S:74.6%,D:1.6%],F:1.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC:72.7%[S:71.0%,D:1.8%],F:2.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBUSCO eukaryota\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;255)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC:91.0%[S:90.2%,D:0.8%],F:2.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC:90.2%[S:85.5%,D:4.7%],F:2.4%,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC:89.4%[S:88.2%,D:1.2%],F:2.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC:87.1%[S:86.3%,D:0.8%],F:3.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparative Genomics and Orthogroups\u003c/h2\u003e \u003cp\u003eThe BRAKER pipeline predicted a total of 15,875 protein-coding genes in the \u003cem\u003eC. annulatus\u003c/em\u003e genome. The OrthoFinder analysis clustered 58,355 proteins from the four tardigrade species into 13,419 orthogroups. A Venn diagram illustrating the distribution of shared and unique orthogroups is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e (see detailed list of orthogroups in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A key outcome is the identification of 318 orthogroups unique to \u003cem\u003eC. annulatus\u003c/em\u003e. These unique orthogroups collectively contain 1441 \u003cem\u003eC. annulatus\u003c/em\u003e proteins, of which 719 received eggNOG functional annotations (COG/Pfam/KO fields available) and were used for quantitative functional profiling (see functional annotation of \u003cem\u003eC. annulatus\u003c/em\u003e proteins in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of specific proteins of\u003c/b\u003e \u003cb\u003eC. annulatus\u003c/b\u003e\u003cb\u003e-specific groups by COG categories\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize specific proteins functions, we compared the COG category composition of eggNOG-annotated proteins from \u003cem\u003eC. annulatus\u003c/em\u003e-unique orthogroups (n\u0026thinsp;=\u0026thinsp;719) against the full set of eggNOG-annotated proteins in the \u003cem\u003eC. annulatus\u003c/em\u003e proteome (n\u0026thinsp;=\u0026thinsp;12,042). In the unique set, the most represented categories were T (signal transduction mechanisms; 259 prot., 36.0%), O (posttranslational modification, protein turnover, chaperones; 119, 16.6%) and S (function unknown; 110 prot., 15.3%). Additional contributions were observed for G (carbohydrate transport and metabolism; 39), P (inorganic ion transport and metabolism; 32) and C (energy production and conversion; 29), whereas core \u0026ldquo;housekeeping\u0026rdquo; functions, such as translation-related J, were underrepresented among unique proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Overall, \u003cem\u003eC. annulatus\u003c/em\u003e-specific orthogroups show a pronounced shift toward regulatory/sensory functions (COG T) and protein homeostasis modules (COG O), together with a substantial fraction of proteins that remain functionally uncharacterized (COG S).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePfam domain enrichment among\u003c/b\u003e \u003cb\u003eC. annulatus\u003c/b\u003e\u003cb\u003e-unique orthogroup proteins\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePfam profiling of proteins from unique orthogroups revealed clear domain-level signatures consistent with the COG-based shift toward signaling and extracellular functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The most frequent domains among unique proteins were: Pkinase (185), Pkinase_Tyr (131), ANF_receptor (120), Guanylate_cyc (116), HNOBA (109). This combination is characteristic of receptor-type guanylate cyclase architectures and strongly suggests a species-specific expansion of cGMP-linked receptor signaling in \u003cem\u003eC. annulatus\u003c/em\u003e. Additional enriched domains included 7tm_1 (43) (GPCR-like receptors), and multiple extracellular/ECM-associated modules: Astacin (68), TSP_1 (53), EGF (48), Trypsin (45), ShK (42), CUB (40), as well as Lectin_C (35) and BTB (29). Collectively, these data indicate that the unique orthogroup fraction is dominated by coherent expansions of sensory signaling receptors and extracellular remodeling proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further inspect lineage-specific proteins lacking functional assignments, we focused on the subset classified as COG category S (Function unknown) within the \u003cem\u003eC. annulatus-\u003c/em\u003eunique orthogroups. Among these 110 corresponding proteins, all contained identifiable Pfam domains, indicating that they represent structurally defined proteins that included recurrent signatures associated with membrane-associated signaling (e.g., 7tm_1 and XK-related domains), chromatin and transcriptional regulation (e.g., Chromo, SET, PHD and zinc-finger domains), extracellular interactions (e.g., EGF, Lectin_C and CAP domains). Together, these results suggest that the \u0026ldquo;function unknown\u0026rdquo; proteins likely represent species-specific regulatory and signaling components contributing to \u003cem\u003eC. annulatus\u003c/em\u003e-specific adaptations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStress-response and repair-related candidates\u003c/h2\u003e \u003cp\u003eSeveral unique orthogroups contain proteins classically implicated in stress tolerance and cellular repair (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e): antioxidant enzymes (SOD-like), chaperones (HSP70-like), selective autophagy receptors (NBR1-like), ubiquitin-mediated proteostasis regulators (BTB/Kelch adaptors), and DDR-associated ubiquitin ligases (e.g., HUWE1-like). Notably, HSP70-family chaperones are generally expected to be conserved; therefore, their assignment to a \u003cem\u003eC. annulatus\u003c/em\u003e-specific orthogroup likely reflects orthogroup splitting and/or divergence of particular paralogs rather than true lineage-specificity. Together, these proteins form a coordinated set of mechanisms that support oxidative stress control, proteome maintenance, and DNA damage response pathways.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eCucumibius annulatus\u003c/em\u003e-specific orthogroups with stress-response and repair-related proteins\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrthogroup\u003c/p\u003e \u003cp\u003e(#of proteins)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctional relevance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eAntioxidant protection (SOD1 (Superoxide dismutase Cu-Zn) and sod)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0010046\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eKO: K04565; Pfam: \u003cb\u003eSod_Cu\u003c/b\u003e. Canonical antioxidant enzyme implicated in reactive oxygen species (ROS) detoxification and commonly discussed in the context of stress tolerance.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0009925\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHSP70-like putative paralogs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0011604\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSP70 is a central molecular chaperone that maintains proteostasis, particularly under stress, by binding unfolded or misfolded proteins, promoting their proper folding, and preventing aggregation.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSelective autophagy / protein quality control\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0001225\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePresence of NBR1 (selective autophagy receptor; \u0026ldquo;next to BRCA1 gene\u0026rdquo;), with domain features consistent with PB1/ZZ/UBA modules. Supports a role in stress-associated clearance of damaged proteins/organelles.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUbiquitination / BTB\u0026ndash;Kelch (regulators of protein degradation)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0000257\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(24)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnriched in BTB/BACK/Kelch proteins, which often act as CUL3 E3 ligase adaptors. Expansion may indicate lineage-specific remodeling of ubiquitin-mediated proteostasis and stress signaling.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE3 ubiquitin ligases (stress/DDR regulation)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0010267\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRFFL/FEM1-like E3 ubiquitin ligases with ankyrin repeats; regulatory proteins potentially involved in stress and DNA damage response (DDR) pathways\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDNA repair\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0011414\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOne protein is annotated as HUWE1 (HECT E3 ubiquitin ligase), which has reported links to ubiquitin-mediated regulation of DNA damage response and/or repair-associated processes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChitin/cuticle-related proteins potentially involved in cuticle remodeling\u003c/h2\u003e \u003cp\u003eA second set captures candidates potentially involved in tardigrade cuticle organization and remodeling (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), including a canonical GH18 chitinase with CBM14 (OG0011702), multiple CBM14 chitin-binding proteins (Peritrophin-A-like signals), multidomain chitin-associated proteins (e.g., CBM14\u0026thinsp;+\u0026thinsp;LysM\u0026thinsp;+\u0026thinsp;polysaccharide deacetylase-like combinations), and extracellular proteases with chitin-binding modules. This repertoire is consistent with the observed enrichment of extracellular domains (TSP_1/EGF/CUB/Trypsin/Astacin) and supports the hypothesis of a dynamically regulated and lineage-specific extracellular/cuticular toolkit in \u003cem\u003eC. annulatus\u003c/em\u003e. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChitin-related proteins potentially involved in cuticle organization and remodeling in \u003cem\u003eCucumibius annulatus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrthogroup\u003c/p\u003e \u003cp\u003e(#of proteins)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePfam / domains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKO / annotation clue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRole in tardigrade cuticle biology\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eChitinases (chitin degradation / remodeling)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0011702\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlyco_hydro_18\u0026thinsp;+\u0026thinsp;CBM_14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKO: K01183 (chitinase)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCanonical GH18 chitinase with a chitin-binding module; likely involved in remodeling of chitin-containing structures (e.g., cuticle)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eChitinase-like (atypical domain architecture; requires validation)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0010212\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlyco_hydro_19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChitinase class I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChitinase-like protein; GH19 is unusual for animals\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0011447\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eChitin-binding proteins (structural/extracellular components)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0001235\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCBM_14; sometimes Chitin_bind_4 (e.g., g15380.t1; g1810.t1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeritrophin-A / chitin binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCandidate chitin-binding extracellular/cuticular proteins that may contribute to cuticle architecture and mechanical properties\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eChitin binding\u0026thinsp;+\u0026thinsp;chitin metabolic process (multidomain)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0011703\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCBM_14, LysM, Polysacc_deac_1 (+\u0026thinsp;Cu-binding_MopE)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echitin binding; chitin metabolic process\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMultidomain candidate involved in chitin-associated metabolism or matrix interactions; may modulate chitin structure (e.g., via polysaccharide deacetylase-like activity)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProteases with chitin-binding modules (extracellular remodeling)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0000353\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(19)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin_bind_4, NIDO, Somatomedin_B, Trypsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprotease-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSecreted/ECM-associated protease architecture with chitin-binding features; potentially participates in cuticle/ECM remodeling (protein component turnover and matrix processing)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProtease with CBM_14 (candidate cuticle remodeling factor)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0011230\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCBM_14, Trypsin (+\u0026thinsp;PAP2, RED_C/RED_N, RRM_1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprotease-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCandidate proteolytic component with chitin-binding module; may contribute to remodeling of cuticle-associated proteins (domain combination suggests complex regulation)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eChitin synthase\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0007650 (1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChitin_synth_1,Chitin_synth_1N,Chitin_synth_2,EGF,F5_F8_type_C,FYVE_2,Glyco_hydro_47,Glyco_trans_2_3,I-set,JAB,Myosin_head,OTU,PKD_channel,Rab_eff_C,SAM_1,SAM_2,SRCR,TSP_1,UQ_con\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eko:K00698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eWithin two orthogroups, they are also found in other species:\u003c/p\u003e \u003cp\u003ervar (1 ), parmetro (3), hypexem (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eOG0003428 (2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLineage-specific expansions and molecular adaptations\u003c/h2\u003e \u003cp\u003eExtensive expansion of receptor-associated signaling proteins in \u003cem\u003eC. annulatus\u003c/em\u003e genome suggest that it invests heavily in membrane-associated sensing and regulatory networks, potentially enhancing the detection of environmental changes and coordination of physiological transitions associated with dehydration and cryptobiosis. A second major lineage-specific theme is the expansion of extracellular and matrix-associated proteins. Together with the presence of chitin- and cuticle-related proteins (including GH18 chitinases with CBM14 and multiple chitin-binding proteins), these findings suggest that \u003cem\u003eC. annulatus\u003c/em\u003e possesses an expanded toolkit for cuticle remodeling and extracellular structural maintenance, processes that are critical during molting and may contribute to mechanical stability and barrier function during desiccation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCucumibius annulatus\u003c/em\u003e is the first species of Isohypsibioidea for which a genome has been assembled. Based on rRNA tree topology, this superfamily is a basal lineage within Parachela [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eC. annulatus\u003c/em\u003e is strictly freshwater, does not occur in terrestrial habitats, and most likely cannot tolerate drying out. Phylogenetic placement raises the possibility that the prominent expansions we observe\u0026mdash;especially in receptor-like signaling proteins and chitin/cuticle-associated genes\u0026mdash;reflect either retained ancestral features or lineage-specific adaptations linked to the unusually complex cuticle morphology of \u003cem\u003eC. annulatus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eWe additionally identified a lineage-specific expansion of globin-domain proteins (OG0000357; 19 proteins). Beyond classical roles in oxygen transport, globins in small invertebrates are often implicated in redox balance and stress physiology. In the context of cryptobiotic cycles and fluctuating oxygen availability, an expanded globin repertoire may contribute to oxygen homeostasis and mitigation of oxidative stress during entry into and recovery from dormant states.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConserved maintenance systems versus lineage-specific solutions\u003c/h2\u003e \u003cp\u003eDespite the pronounced lineage-specific expansions, \u003cem\u003eC. annulatus\u003c/em\u003e retains a conserved core of stress-response and maintenance pathways typical for tardigrades. Comparative analysis revealed the presence of key DNA repair enzymes (including XPF-, MUS81-, FEN1-, DNA2-, and EXO1-like proteins), as well as a well-developed RNA surveillance and RNAi machinery (exosome components, mRNA decapping and degradation factors, and Dicer- and MUT-7-like proteins), supporting an active model of genome and transcriptome maintenance under stress.\u003c/p\u003e \u003cp\u003eImportantly, \u003cem\u003eC. annulatus\u003c/em\u003e shares canonical tardigrade heat-soluble protein families with \u003cem\u003eR. varieornatus\u003c/em\u003e, including 16 CAHS, 13 SAHS and 3 LEA proteins, indicating that the classical stress-protection toolkit is conserved and may support basal stress tolerance. At the same time, the retention of CAHS, SAHS (and LEA) may be biologically relevant beyond anhydrobiosis, as CAHS proteins have been implicated in protection against multiple stress types, including cold stress/cryobiosis. This is consistent with the distribution of \u003cem\u003eC. annulatus\u003c/em\u003e in boreal and mountainous regions (so called Arctic-alpine areal type) where freshwater habitats can freeze extensively. In contrast, we did not detect convincing Dsup-like homologs in the predicted \u003cem\u003eC. annulatus\u003c/em\u003e proteome using similarity searches with Dsup from \u003cem\u003eR. varieornatus\u003c/em\u003e, suggesting that a Dsup-based DNA-shielding mechanism is either absent or too divergent to be recovered by homology-based approaches [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, we detect convincing candidates for the trehalose biosynthesis machinery (TPS\u0026ndash;TPP) in the predicted \u003cem\u003eC. annulatus\u003c/em\u003e proteome\u0026thinsp;\u0026minus;\u0026thinsp;9 proteins with trehalose phosphatase domains (Trehalose_PPase). This number is consistent with the amount of trehalose biosynthesis pathway members among other tardigrade species in the study. Trehalose can act as a cryoprotectant and chemical chaperone. Overwintering under ice can impose hypoxic conditions. The lineage-specific expansion of globin-domain proteins in \u003cem\u003eC. annulatus\u003c/em\u003e therefore may be interpreted not only in terms of oxidative stress control, but also as a potential adaptation supporting oxygen homeostasis during seasonal oxygen limitation.\u003c/p\u003e \u003cp\u003eHowever, in contrast to \u003cem\u003eR. varieornatus\u003c/em\u003e, the dominant lineage-specific trend in \u003cem\u003eC. annulatus\u003c/em\u003e is not the continued expansion of the heat-soluble proteins, but rather large-scale diversification of signaling receptors and extracellular proteins. These results suggest that \u003cem\u003eC. annulatus\u003c/em\u003e achieves extremotolerance through a combination of conserved protective mechanisms and lineage-specific regulatory and structural adaptations. In particular, the expansion of signaling receptors and extracellular/structural protein families distinguishes \u003cem\u003eC. annulatus\u003c/em\u003e from other sequenced tardigrades and may represent an important, previously underappreciated component of tardigrade adaptation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we provide the first genome assembly, structural annotation and comparative orthogroup-based analysis for the recently established tardigrade genus \u003cem\u003eCucumibius\u003c/em\u003e and its species \u003cem\u003eCucumibius annulatus\u003c/em\u003e. The genome-wide comparison against three reference tardigrades indicates 318 orthogroups unique to \u003cem\u003eC. annulatus\u003c/em\u003e species, with a functional profile strongly shifted toward signal transduction and protein turnover/proteostasis. This pattern suggests that \u003cem\u003eC. annulatus\u003c/em\u003e combines a conserved \u0026ldquo;core\u0026rdquo; of stress-response mechanisms with pronounced lineage-specific expansions that likely affect how environmental cues are sensed and how extracellular structures are maintained.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed with the content and that all gave explicit consent to submit. They obtained consent from the responsible authorities at the institute where the work has been carried out\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome assembly was deposited as NCBI BioSample, accession number SAMN55414403\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Sample collection and material preparation were performed by Denis Tumanov. Preparation of material for library construction and sequencing was performed by Elena Nassonova. Data collection and analysis were performed by Daria Makarova and Mikhail Rayko. The first draft of the manuscript was written by Daria Makarova and all authors commented on previous versions of the manuscript. \u0026nbsp;All authors read and approved the final manuscript. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Russian Science Foundation: project no. 25-74-20033 (sample collection, morphological description, and species identification) and project no. 23–74-00071 (DNA extraction, whole genome amplification, sequencing, metagenome assembly, analysis of parasite and host genomes, bioinformatic data processing).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This study utilised equipment of the Core Facility Centres ‘Biobank’, ‘Development of Molecular and Cell Technologies’ and ‘Culturing of microorganisms’ of the Research Park of Saint Petersburg University\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuidetti R, J\u0026ouml;nsson KI (2002) Long-term anhydrobiotic survival in semi-terrestrial tardigrades. 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Mol Biol Evol 34(8):2115\u0026ndash;2122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msx148\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msx148\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"genome, tardigrade, stress response, comparative genomics","lastPublishedDoi":"10.21203/rs.3.rs-8958382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8958382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTardigrades, or water bears, are microscopic invertebrates renowned for their ability to survive extreme environmental conditions through a process called cryptobiosis. The genomic underpinnings of this remarkable resilience are a subject of intense scientific interest. In this study, we present the first genome assembly and annotation of a tardigrade species \u003cem\u003eCucumibius annulatus\u003c/em\u003e, recently redescribed from North-West Russia. The draft genome was assembled de novo using DNBSEQ sequencing data (PE300 reads), followed by decontamination and annotation. Final assembly has a size of 73 Mb and a BUSCO completeness score of 87.1%. Comparative genomic analysis against three other tardigrade species (\u003cem\u003eHypsibius exemplaris\u003c/em\u003e, \u003cem\u003eRamazzottius varieornatus\u003c/em\u003e, and \u003cem\u003eParamacrobiotus metropolitanus\u003c/em\u003e) revealed unique orthologous gene groups in \u003cem\u003eC. annulatus.\u003c/em\u003e We identified significant expansions in protein families associated with stress response, including antioxidant enzymes (e.g., Superoxide Dismutase), chaperones (HSP70), and components of the DNA repair and ubiquitin-proteasome systems. Notably, \u003cem\u003eC. annulatus\u003c/em\u003e exhibits a pronounced species-specific expansion of signaling receptors, particularly receptor guanylate cyclases and G-protein coupled receptors, as well as extracellular matrix proteins like metalloproteases. These findings suggest that adaptations in signaling pathways and structural proteins, in addition to typical stress-response genes, may play a crucial role in the survival strategies.\u003c/p\u003e","manuscriptTitle":"Genome of the tardigrade Cucumibius annulatus: functional annotation and specific proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 14:41:42","doi":"10.21203/rs.3.rs-8958382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-11T04:48:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-08T14:30:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97528525282311037434552893817324845354","date":"2026-03-06T04:11:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-03T11:35:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T01:12:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-26T01:12:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2026-02-24T13:55:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d8c0ecda-1d0f-4704-a024-7acf1d27c984","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-11T04:53:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 14:41:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8958382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8958382","identity":"rs-8958382","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}